Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls.

A steroidal compound was recently detected in a seized black market product and was identified as (17α,20E)-17,20-[(1-methoxyethylidene) bis (oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11). This compound is described to possess selective androgen receptor modulator- and myostatin inhibitor-like properties. As YK11 is an experimental drug candidate and a non-approved substance for humans, scientific data on its metabolism is scarce. Due to its steroidal backbone and the arguably labile orthoester-derived moiety positioned at the D-ring, substantial metabolic conversion in vivo was anticipated. To unambiguously detect urinary metabolites of YK11, an elimination study with six-fold deuterated YK11 was conducted. Post-administration specimens were analyzed using hydrogen isotope ratio mass spectrometry coupled to single quadrupole mass spectrometry to identify metabolites alongside basic mass spectrometric data. Further characterization of those metabolites relevant to sports drug testing was accomplished using gas chromatography-high resolution-high accuracy mass spectrometry. Fourteen deuterated urinary metabolites were detected comprising unconjugated, glucuronidated, and sulfoconjugated metabolites. As expected, no intact YK11 was observed in the elimination study urine samples. While the unconjugated metabolites disappeared within 24 hours post-administration, both glucuronidated and sulfated metabolites were traceable for more than 48 hours. The chemical structures of the two most promising glucuronidated metabolites (5ß-19-nor-pregnane-3α,17ß,20-triol and 5ß-19-nor-pregnane-3α,17ß-diol-20-one) were verified by in-house synthesis of both metabolites and confirmed by nuclear magnetic resonance analysis. In order to elucidate their potential in sports drug testing, both were successfully implemented into the currently applied analytical method for the detection of anabolic agents.

Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry.

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd.

The atypical excretion profile of meldonium: Comparison of urinary detection windows after single- and multiple-dose application in healthy volunteers.

Following a one-year monitoring program providing unequivocal analytical evidence for a high prevalence in international elite sports, meldonium has been included in the World Anti-Doping Agency's (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-dose (5 volunteers, 1×500mg) and multiple-dose oral application (5 volunteers; 2×500mg/day for 6days) were determined in order to facilitate the result management concerning meldonium findings in doping controls. Particularly the option to differentiate between recent use and tapering concentrations was studied. Urinary meldonium concentrations were determined using an analytical approach based on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after oral application of meldonium and demonstrates that urinary detection windows are of considerable extent with up to 65 and 117days (concentrations>LOQ of 10ng/mL) following single- and multiple-dose applications, respectively.

Periostin gene variants are associated with disease course and severity in juvenile idiopathic arthritis.

OBJECTIVES: This study aimed to identify polymorphic variants of the Periostin gene associated with disease severity and clinical course in children with juvenile idiopathic arthritis. METHODS: DNA genotyping of 7 single-nucleotide polymorphisms within the periostin gene was performed in 117 patients and their parents and in 102 control samples. Our patients were divided in the following 4 disease categories: 1) persistent oligoarthritis; 2) extended oligoarthritis; 3) polyarthritis; 4) systemic arthritis. Quantitative association analysis was performed in order to test for association between the 7 genetic variants and 18 selected clinical traits. RESULTS: A harmful association was observed between the minor allele of rs17197936 and 2 clinical traits, count of joints with active arthritis and count of joints with pain on motion/tenderness, in patients with extended oligoarthritis. Furthermore, the haplotype represented by the minor allele variants of rs3829364, rs6750 and rs9547951 showed an unfavourable association with the above 2 traits plus the following 3 in the whole patient group: juvenile arthritis damage index articular score, childhood health assessment questionnaire score and disease duration. CONCLUSIONS: These associations suggest that the variants involved can be regarded as genetic factors influencing some phenotypic aspects of juvenile idiopathic arthritis. Genotyping of this gene may represent a useful tool to identify patients who are at greatest risk of experiencing a poorer long-term outcome.

Use of dried blood spots in doping control analysis of anabolic steroid esters.

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters. The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Ultrahigh pressure liquid chromatography-(tandem) mass spectrometry in human sports drug testing: possibilities and limitations.

Doping control analytical laboratories for human sports predominantly employ nowadays chromatographic-mass spectrometric test methods for routine, high throughput screening and confirmation assays concerning low and high molecular mass analytes. Liquid chromatography-(tandem) mass spectrometry [(LC-MS(/MS)] and particularly ultrahigh pressure liquid chromatography (UHPLC)-MS/MS instruments have become devices of choice due to their indispensable capabilities that compensate for limitations inherent to other commonly used strategies such as immunological and gas chromatography-(tandem) mass spectrometry [(GC-MS(/MS)]-based detection methods. UHPLC-MS/MS-based assays at low mass spectrometric resolution have been established allowing for fast and sensitive targeted analyses focusing on pre-selected target analytes with diagnostic precursor-product ion pairs. Combining UHPLC to high resolution/high accuracy MS(/MS) further expanded the targeted approach (i.e., plotting extracted ion chromatograms of protonated or deprotonated molecules as well as product ions measured with accurate masses) toward non-targeted analyses enabling also retrospective data mining. In this review, recent applications of UHPLC-MS/MS in sports drug testing procedures published between 2008 and 2012 are presented and advantages as well as limitations in a short- and long-term perspective are discussed.